Saccharomyces cerevisiae pyruvate kinase Pyk1 is PKA phosphorylation substrate in vitro

Fractionation of Saccharomyces cerevisiae postribosomal extract on DEAE-cel lulose revealed two fractions of cAMP-dependent protein kinase (PKA-1 and P KA-2). The presence of PKA in both fractions was confirmed by immunoblottin g with anti-Bey I antibodies. Yeast pyruvate kinase Pyk1 identified by amin o acid microsequencing analysis and immunoblotting with anti-Pyk1 antibodie s copurified with the PKA-I but not the -2 fraction. Pyk1 can be phosphoryl ated by yeast PKA in vitro in the presence of cAMP and cGMP. Two-dimensiona l gel electrophotetic analysis revealed four phosphorylated forms of Pyk1 m odified by PKA. In phosphorylation of Pyk1 mainly the Tpk2 catalytic subuni t of yeast PKA was involved. (C) 2001 Federation of European Microbiologica l Societies. Published by Elsevier Science BN. All rights reserved.